Re: [ccp4bb] [OFF-Topic] GST-tagged protein refold protocol

[Rafael Marques]

Hi Dave, 

I should have mentioned that, but yes, I tried 16 degrees overnight and even up to 40 hours. The _expression_ level is great, majority is in the pellet though and the elution fraction is far from desirable for my kinetic assays. 

Best wishes 

______________________________________________________


Rafael Marques da Silva

PhD Student – Structural Biology

University of Leicester


Mestre em Física Biomolecular

Universidade de São Paulo


Bacharel em Ciências Biológicas

Universidade Federal de São Carlos


phone: +44 07861 273773 


           "A sorte acompanha uma mente bem treinada"

On 11 Mar 2025 13:16, David Briggs <david.bri...@crick.ac.uk> wrote:

Hi Rafael, 

I have you tried reducing the temperature post-induction?

You can grow the cells to OD600 <whatever>, reduce the temperature (I have gone as low as 16ºC), add IPTG and harvest the cells the following morning. 

The reduction in temperature can slow down the rate of protein production allowing the nascent polypeptide chain more time to fold correctly. 

(At least this is how I rationalise the observed increased in folded soluble protein in my head)

I would certainly play with the temperature before going down the refolding route. 

Good luck,

Dave

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Dr David C. Briggs CSci MRSB (he/him)

Principal Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

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From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Rafael Marques <rafael_mmsi...@hotmail.com>
Sent: 11 March 2025 11:18
To: CCP4BB@JISCMAIL.AC.UK <CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] [OFF-Topic] GST-tagged protein refold protocol

 

 

External Sender: Use caution.

 

Hello folks, 

I have been trying to purify a GST-tagged protein for a while but the results are far below the expected. The majority of my sample is in the pellet fraction and the yield of my _expression_ is very good. This is known because the protein was identified both by Mass Spectrometry and Western-blot. I believe my protein is laying inside inclusion bodies. I have tried to diminish the IPTG concentration, express it for a shorter time and also different E.coli strains but the results are still the same. As a last resort, I was thinking about refolding. I found this paper (attached), from 2004, but I was wondering if someone who has experience regarding protein refolding could shed some light on this topic, principally if there are more updated protocols. 

Best wishes

P.S. His-tag did not improve my results too

______________________________________________________

Rafael Marques da Silva

PhD Student – Structural Biology

University of Leicester

Mestre em Física Biomolecular

Universidade de São Paulo

Bacharel em Ciências Biológicas

Universidade Federal de São Carlos

phone: +44 07861 273773 

           "A sorte acompanha uma mente bem treinada"

________________________________________________

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